Method, kit and system for collecting and processing samples for DNA and RNA detection
View Patent ↗This invention relates to a method, kit and system for collecting and processing of samples to release and treat DNA and RNA for gene sequence detection. The method described here in provides for rapid and convenient release, and recovery of DNA and RNA from tissues and cellular materials.
1. A method for collecting, processing, and analyzing a target nucleic acid from a sample comprising:
(a) providing a sample wherein the sample is a shrimp tissue comprising nucleic acids, wherein the target nucleic acid is diagnostic of a shrimp pathogen-comprising white spot syndrome virus;
(b) transferring the sample to a flexible-walled vessel, which can be inverted so that the exterior wall becomes the interior wall and in which sample materials can be agitated and suspended by applying and releasing external pressure to the walls;
(c) adding an extraction fluid to the vessel;
(d) processing the sample with the extraction fluid and one or more treatment reagents in the vessel to release nucleic acid from the sample to solubilize the nucleic acid and produce a recovery fluid;
(e) filtering the recovery fluid produced in step (d) wherein the vessel contains filter media to remove particulates; and
(f) transferring a portion of the recovery fluid or product from step (e) to a suitable detection system for detection of the target nucleic acid, wherein the detection system for detection of the target nucleic acid uses PCR or RT-PCR for amplification of the target nucleic acid using diagnostic primer sequences for the shrimp pathogen, and wherein the diagnostic primer sequences for the white spot syndrome virus are selected from: (i) SEQ ID NO: 1 and SEQ ID NO: 2; (ii) SEQ ID NO: 3 and SEQ ID NO: 4; (iii) SEQ ID NO: 5 and SEQ ID NO: 6; and (iv) SEQ ID NO: 7 and SEQ ID NO: 8.
2. The method of claim 1 wherein the sample comprises a solid material and prior to step (c) the process comprises a step of crushing the sample in the flexible-walled vessel and the processing step (d) comprises mixing and macerating the sample with the extraction fluid and treatment reagent(s) in the vessel to aid in release of the nucleic acid.
3. The method of claim 1 wherein the one or more treatment reagents comprises a lysing agent, which comprises a detergent, an enzyme, or a combination thereof.
4. The method of claim 3 wherein the one or more treatment reagents further comprises a buffer having pH in the range of 7 to 7.8, magnesium chloride, and potassium chloride.